Pertussis vaccine preparation



PERTUSSlS VACCINE PREPARATION Otto K. Behrens and Paul W. Ensminger,Indianapolis, Ind., assignors to, Eli Lilly and Company, Indianapolis,Ind., a corporation of Indiana No Drawing. Application October 23, 1952Serial No. 316,566

7 Claims. (Cl. 167-78).

:IEhlS invention relatesto an antigen-derived from H emophiluspertussis, and to the process for its preparation.

Whooping cough is a serious disease of infancy, responsible for numerousfatalities in unimmunized children. It has heretofore been possible toimmunize children against whooping cough by the use of-vaccines. Suchvaccines commonly consist of a suspension of killed Hemophilus pertussisorganisms in saline alone, or in saline and alum, the latter being knownas alum-precipitated vaccine. Those vaccines, While eflicacious, are notwithout certain inherent disadvantages. For example, their use hasresulted in numerous cases of sterile abscesses among those immunized.Furthermore, toxic symptoms, as evidenced by fever and irritation of thetissues after the vaccine has been injected, are "common side reactions,even where necrosis or other severe inflammatory reactions do not occur.In some instances, encephalitis has resulted from immunization with theknown Vaccines.- q

It is an object of this invention to provide an antigen derived fromHemophilus pertussis cells which is substantially non-irritating to thetissues into which it is administered, yet which is capable of providinga lasting immunity in human beings. It is another object of thisinvention to provide a preparation capable of evoking an immunityagainst whooping cough. his a further object of this invention toprovide an antigen which is characterized by the absence therefrom ofbacterial toxins, cells and debris. Other objects will be apparent fromthe disclosures made hereinafter.

In accordance with the above and other objects, we have discovered thata useful antigen for immunization against Whooping cough, havingextremely low toxicity both. systemically and locally can be prepared byextracting intact Hemophilus pertussis cells with an aqueous solutioncontaining a controlled amount of a phenolic compound.

In'broad outline, the manner of carrying out this invention forpreparing pertussis vaccine is as follows:

' Hemophilus pertussis is cultured upon agar or charcoal-agar media inaccordance with the usual methods employed for the preparation ofpertussis vaccine, and the bacterial growth is harvested by washing thebacterial cells from the agar growth medium with water or salinesolution. To the suspension of bacterial cells is added a controlledamount of. a phenolicv compound, and the-cells are allowed. to stand incontact with thesolution of the phenolic compound for from one to threeweeks during which time the antigenic material is extracted from thecells. The mixture is shaken from time to time to assist theextraction,the shaking being carried out sufiiciently gently so that the cells willnot be disrupted.

' The intact cells areseparated from the extraction sol- Yen't as bycentrifugation or filtration, leaving a centrifus Patent gate orfiltrate comprising the antigenic material dissolved in the aqueoussolution of the phenolic compound.

In cases where the aqueous phenolic solution has a phenolicconcentration greater than about 0.5 percent, the concentration later isreduced to an amount not greater than about 0.5 percent, to provide avaccine which can be administered parenterally Without untoward efie-ctscaused by the phenolic compound. Reduction can be secured by anysuitable procedure, for example, by solvent extraction, or by dialysis.Throughout the process, aseptic conditions should be observed as isstandard practice in the preparation of vaccines.

The antigen-containing solution thus produced is a clear, colorlessliquid having an antigenic potency which is'proportional to the numberof cells present in the origi nal bacterial suspension. The solution isstandardized by assay procedures of the National Institutes of Health,which consist of the injection into mice of the antigenic material withthe production of a typical immunity against challenge doses of virulentpertussis organisms. After standardization, the antigenic solution canbe brought to the strength desired by dilution or by evaponation invacuo at room temperature to provide a vaccine of desired potency. Thevaccine can also if desired, be treated with alum in accordance withcustomary procedures to give an alum-precipitated vaccine. The vaccines,whether clear or alum-precipitated are filled under aseptic conditionsinto dosage containers suitable for diserally produce no reactions. Atthe most, only mild reactions have ever been noted in animal and humanclinical tests. v

The absence of side effects after administration of the vaccinesprepared as described herein are shown in the following table in whichare set forth the results of comparative intradermal skin tests on redrabbits. The tests employ pertussis antigen consisting of cellularmaterial prepared in accordance with conventional procedure, andcell-free pertussis antigen prepared in accordance with this invention.the material employed in the test as set forth in the data below thetable. In each instance 0.2 ml. of the preparation was injectedintradermally. The second column indicates the total number of cellsinjected. It should be noted that with lots B, C, D, and E no cells wereinjected, the cell numbers used in connection with these being 'theconcentration of cells in each ml. of the original suspension which wasextracted. The third col: umn indicates the A; U. (antigenic-units)injected, this figure being determined by the mouse test method of theNational Institutes of Health. The fourth, fifth and sixth columnsrepresent respectively the results of the observations for unfavorablereactions after administration of the antigen, the observations beingmade respectively 24, 48 and 72 hours after the intradermal injection ofthe test animals. The areas of redness and necrosis observed weremeasured. I As used herein, the term A. U. is an expression of theantigenicity of the vaccine as compared with that of the standardvaccine provided by the National Institutes at Health. That standardemploying 20 b llion cells per ml. of vaccine has'an' A. U. value of 20.

In the table, the first column indicates- TABLE Cells injected A. U. in-Skin reaction Lot No. per skin test jected per area skin test area 24Hr. 48 Hr. 72 Hr.

l0. 2 Raised, Red 9 x 11 mun..- Raised, Red 9 x mm., Raised, Red 9 x 10mm,

Necrosis 3 x 3 mm. Necrosis 3 x 3 mm. 5. i Raised, Red 9 x 9 mm. Raised,Red 9 x 7 mm.. Raised, Red 9 x 7 mm. 2. 55 Raised, Red 4 x 5 mm..Raised, Red 6 x 8 mm. Raised, Red fi x 8 mm. 1.28 Red3x3mm RedfixfimmRed5x5mm. 0.64 Red2x2mm Red3x3mm. 0.32 Negative Red 2 x 2 mm Red 2 x 2mm. 0. do Slightly Red Slightly Red. 0. 80 do Negative" Negative. Do.Do. Do. Do.

Lot A: A commercial vaccine containing cell material together withsodium ethyl mercuri thlosalicylate 1l0,000-and formaldehyde 1:1000.

ot B: A vaccine prepared by extraction of cells with 0.2 percent aqueousphenol at 4C. for 14 days. Lot 0: A vaccine prepared by extraction ofcells with 0.2 percent aqueous m-cresol at 4 C. or 14 days. Lot D: Avaccine prepared hy extraction of cells with 0.2 percent aqueous phenolat 25 C. for 14 days. Lot E: A vaccine prepared by extraction of cellswith 0.2 pcrccnt aqueous m-cresol at 37 C. for 14 days.

In employing the extraction process of this invention, the extractionstep is carried out at a temperature betweenabout 4 C. and about 40 C.The most efiicient extraction temperature appears to be about 37 C. Atthat temperature, extraction of antigenic material is substantiallycomplete after about 14 days, and the vaccine prepared from the extractis substantitally free from materials which cause side effects. Increaseof the extraction temperature above 40 C. has an unfavorable effect onthe quantity and quality of the vaccine produced.

Numerous phenolic compounds can be employed to furnish the phenoliccontent of'thc aqueous extraction solvent. Among the phenolic compoundswhich provide the most satisfactory results, both with respect to theantigenie potency and the freedom from irritating substances of thevaccines produced, are the cresols, the mononitrophenols, phenol, andcarvacrol. Other examples of phenolic compounds which can be usedinclude the chlorophc'nols, hydroquinone, catechol, resorcinol,2,4-dinitrophenol, pyrogallol, picric acid, phloroglucinol, and thelike. Additional phenolic compounds suitable for the purposes of thisinvention will readily be apparent.

To provide effective extraction of the antigenic material but notextraction of irritating substances, the amount of phenolic compoundemployed in the aqueous extraction solvent should be from about 0.05 toabout 2.0 percent, on a weight-volume basis. Concentrations above theupper limit have an increasingly destructive effect upon the antigen,and concentrations below the lower limit have but little extractiveability. A concentration of "about 0.2 percent is preferred since thatconcentration provides efficient extraction, but yet is below the upperpermissible concentration in the final vaccine. Moreover, such aconcentration of phenolic content is desirable for preservativepurposes.

The following examples further illustrate this invention.

Example 1 Ten liters of charcoal agar were prepared according to theprocedure described by H. M. Powell et 211., Public Health Reports 66,346 (1951), and were placed in twenty toxin bottles containing 500 ml.of media. Each bottle was seeded with 12 ml. of a stock 24-hour smoothculture of Hemophilus pertussis. The seeded bottles were incubated at 37C. for 48 hours, and the organisms thus cultured were washed from thesurface of the charcoal agar in each bottle with 50 cc. of 0.85 percentaqueous sodium chloride. About 1,000 ml. of a suspension of bacterialcells were obtained. The suspension was diluted with 277 ml. of normalsaline solution so that the final c'ell count was about 180billion'cells per ml. To the suspension were added g. of phenol, and themixture teen days, with occasional shaking to resusp'end the cells.After fourteen days, the mixture was centrifuged at about 13,000 R. -P.M. for about one hour, and the supernatant liquid was decanted from thecell sediment. The clear antigen-containing solution thus obtained wastested for potency by injection into mice in accordance with thestandard NIH mouse immunization test.

The clear antigen solution was placed in sterile, cellophane dialysissacks and dialyscd against several changes of pure water over a periodof three days, thereby reducing the phenol concentration to a value ofabout 0.3 percent. The volume increase of the dialysed solution wasremoved by evaporation in vacuo at room temperatrue, and the antigenicpotency of the solution was tested by the NIH procedure. The antigenicsolution contained about 114 A. U. per ml.

Example 2 To 200 ml. of an antigen solution prepared according to theprocedure of Example 1 was added about ml. 0520 percent potassiumaluminum sulfate dodccahydrat'e and 16.6 ml. of 50 percent dibasicsodium phosphate solution. The mixture was allowed to stand about fourhours with occasional shaking and the precipitate containing theantigenic material which formed was separated from the liquid bycentrifugation at about 14,000 R. P. M. The precipitate which containedthe alumprecipitatcd antigenic material was resuspended in 200 ml. ofnormal saline solution, and tested for immunizing potency in miceaccording to the standard test method. The test showed the presence ofabout 63 A. U. per ml.

Example 3 To about 1000 ml. of a suspension of Hemophilus pertussiscells prepared according to the procedure of Example 1 and containingabout 180 billion cells per ml., were added about 2 g. of phenol. Themixture was stored at about 6 C. for about fourteen days, withintermittent shaking to resuspcnd the cells. At the end of fourteendays, the suspension was centrifuged at about 13,000 R. P. M. for aboutone hour and the supernatant liquid, containing the extracted antigen,was subjected to mouse tests for antigenicity. The solutioncontaincd'about 62 A. U. per ml.

Example 4 To about 1000 ml. of a suspension of Hemophilus pertussiscells prepared according to the procedure of Example 1 and containingabout 180 billion cells per ml. were added about2 g. of phenol, and thematerial was stored at about 37 C. for two weeks, during which time thesuspension was gently agitated from time to time to resuspend the cells.The suspension was thereafter cenwas stored at 'a temperatureof about4-6 'C. for fourtrifuged at about l3,000 R. P. M. for about one hourandthe supernatant liquid containing the extracted antigen was subjected tomouse assay tests to determine its immunizing potency. The solutioncontained about 92 A. U. per ml.

Example 5 The procedure of Example 3 was followed, except that 2 g. ofo-cresol were added to the cell suspension.

Upon assay of the antigen solution thus prepared, it was found that theantigen solution contained 130 A. U. per ml.

Example 6 The procedure of Example 3 was followed, but using instead ofphenol 2 g. of mcresol. The cell count of the cell-suspension was about168 billion cells per ml. of suspension.

Mouse assays showed that the resulting antigen solution contained 136 A.U. per ml.

Example 7 The procedure of Example 3 was repeated, except that 2 g. ofo-chlorophenol were added to the cell suspension. Final cell count was168 billion cells per ml.

Assay by standard mouse protection methods showed the presence of 27 A.U. per ml.

Example 8 The procedure of Example 3 was followed, except that 2 g. ofp-nitrophenol were added to the cell suspension. Cells were extracted at168 billion per ml.

Mouse assays showed that the resulting antigen solution contained 12 A.U. per ml.

Example 9 The procedure of Example 3 was repeated, except that 2 g. ofo-nitrophenol were added to the cell suspension. Cells were extracted at168 billion per ml.

Mouse tests showed the presence of 30 A. U. per ml. in the resultingantigen solution.

Example 10 The procedure of Example 3 was followed, except that 2 g. ofcarvacrol were added to the cell suspension.

Mouse assays indicated that the resulting antigen solution containedabout 22 A. U. per ml.

We claim:

1. A method of preparing a reaction-free antigen for immunizationagainst Hemophilus pertussis which comprises extracting intact cells ofHemophilus pertussis for a period of about 1 to about 3 weeks with anaqueous 50 (P- 26113611) 1950, 1

solution of about 0.1 to about 0.2 percent on a weight/ volume basis ofa phenolic compound of the group consisting of phenol, cresol,chlorophenol, nitrophenol, and carvacrol, separating the extract fromthe intact cells, and adjusting the concentration of the phenoliccompound in the separated extraction solution to a value less than about0.5 percent.

2. A method of preparing a reaction-free antigen for immunizationagainst Hemophilus pertussis which comprises extracting intact cells ofHemaphilus pertussis for a period of about 1 to about 3 weeks at atemperature between about 4 C. and about 40 C. with an aqueous solutionof about 0.1 to about 0.2 percent on a weight/ volume basis of aphenolic compound of the group consisting of phenol, cresol,chlorophenol, nitrophenol, and carvacrol, recovering a clear aqueousantigen-containing solution by separating the aqueous phenolic extractfrom the intact cells, and adjusting the concentration of the phenoliccompound to a value less than about 0.5 percent.

3. A method according to claim 2 in which the phenolic compound isphenol.

4. A method according to claim 2 in which the phenolic compound iso-cresol.

5. A method according to claim 2 in which the phenolic compound isp-cresol.

6. The method according to claim 2 in which the phenolic compound iso-nitrophenol.

7. A method according to claim 2 in which the phenolic compound ism-cresol.

References Cited in the file of this patent UNITED STATES PATENTSGerlough Feb. 1, 1944 Pillemer Feb. 1, 1945 OTHER REFERENCES

1. A METHOD OF PREPARING A REACTION-FREE ANTIGEN FOR IMMUNIZATIONAGAINST HEMOPHILUS PERTUSSIS WHICH COMPRISES EXTRACTING INTACT CELLS OFHEMOPHILUS PERTUSSIS FOR A PERIOD OF ABOUT 1 TO ABOUT 3 WEEKS WITH ANAQUEOUS SOLUTION OF ABOUT 0.1 TO ABOUT 0.2 PERCENT ON A WEIGHT/ VOLUMEBASIS OF A PHENOLIC COMPOUND OF THE GROUP CONSISTING OF PHENOL, CRESOL,CHLOROPHENOL, NITROPHENOL, AND CARVACROL, SEPARATING THE EXTRACT FROMTHE INTACT CELLS, AND ADJUSTING THE CONCENTRATION OF THE PHENOLICCOMPOUND IN THE SEPARATED EXTRACTION SOLUTION TO A VALUE LESS THAN ABOUT0.5 PERCENT.